The relative effect of parasite levels, bee population size, and food reserves on winter
mortality and post winter populations of honey bee colonies was estimated. More than 400
colonies were monitored throughout three seasons in Ontario, Canada. Most of the colonies
were infested with varroa mites during the fall (75.7%), but only 27.9% and 6.1% tested
positive to nosema disease and tracheal mites, respectively. Winter colony mortality was
27.2%, and when examined as a fraction of all morbidity factors, fall varroa mite
infestations were the leading cause of colony mortality (associated to > 85% of
colony deaths), followed by fall bee populations and food reserves. Varroa-infested
colonies, with weak populations and low food reserves in the fall, significantly decreased
spring colony populations, whereas varroa infestations and Nosema
infections in the spring, significantly decreased bee populations by early
summer. Overall, results suggest that varroa mites could be the main culprit for the death
and reduced populations of overwintered honey bee colonies in northern climates.

1. INTRODUCTION

Beekeepers, crop growers, authorities, scientists and the general public are all concerned
and alarmed with the mysterious die-offs of honey bee (Apis mellifera)
colonies that have occurred during the last three years in many countries around the world.
The phenomenon has been named colony collapse disorder (CCD) in the USA; many suspects have
been suggested as potential culprits of these losses, but no clear explanation has yet been
found (vanEngelsdorp et al., 2008). So far, no name
or causes have been proposed for the higher than normal winter colony losses beekeepers are
experiencing in Canada (Kevan et al., 2007). In
Ontario, 35% of the province’s colonies died over the winter of 2006–2007 (McRory, 2007), and 32% of them died again during the winter of
2007–2008 (Guzmán-Novoa, 2008). This substantial
number of colony losses is unprecedented in the province and is three times the expected
winter loss during average years.

Although the causes of the record mortality of honey bee colonies remains undetermined,
most scientists agree that it is likely due to a combination of several factors ranging from
viruses, parasites and diseases, to single-source diets, compromised disease resistance,
inclement weather, and pesticides (Stankus, 2008). It
is known for example, that the parasitic mites Varroa destructor and
Acarapis woodi are causes of colony mortality and thus several synthetic
miticides have been used successfully in their control (De Jong, 1997; Wilson et al., 1997; Ellis,
2001). However, mite resistance to the active
ingredients in miticides is now widespread in Europe, the USA and Canada (Elzen et al.,
1998, 1999;
Milani, 1999; Sprefacio et al., 2001; Elzen and Westervelt, 2002;
Thompson et al., 2002; Skinner et al., 2003). Therefore, it is possible that mite populations
are more difficult to control and are causing more damage to colonies in recent years.
Organic miticides such as formic and oxalic acid have been used more in recent years, but it
is possible that these products may not provide enough protection against the
above-mentioned parasitic mites.

High levels of nosema disease (Nosema apis) may also cause colony
mortality during winter or poor colony buildup during spring (Bailey and Ball, 1991). Nosema disease is endemic in honey bee colonies in
Ontario, but its association with colony losses in the province remains unknown.
Additionally, a new Nosema species, Nosema ceranae, was
recently found to infect Apis mellifera colonies (Fries et al., 2006; Huang et al., 2007). The presence of Nosema ceranae was confirmed in Canada in
2007 (Williams et al., 2008). This new species of
Nosema has been linked to the collapse of thousands of colonies in parts
of Europe (Higes et al., 2006, 2007, 2008; Martín-Hernández et
al., 2007) and could likely be a suspect of colony
losses in Ontario. Other causes of winter colony mortality may include starvation due to
insufficient food reserves and weak colony populations during winter (Free and Racey, 1968; Stankus, 2008; vanEngelsdorp et al., 2008).

The objective of this study was to investigate the relative effect of five factors that
presumably could contribute to winter colony mortality and to decreased bee populations in
the spring and summer. Specifically, we looked at low food reserves, low bee population,
nosema disease and infestation of varroa and tracheal mites in commercial honey bee
colonies.

2. MATERIALS AND METHODS

A total of 408 colonies located in six different regions of southern Ontario, Canada, were
randomly selected to assess presumed causes of honey bee mortality and correlate them with
colony seasonal conditions and die offs. The regions where colonies were located included
the counties of Norfolk, Six Nations, Wellington, Grey, Niagara and Middlesex. Selected
colonies belonged to beekeepers that did not apply any disease control procedures during the
duration of the study (late October 2007 to late June 2008). Previous to being selected,
most colonies had been treated with different miticides within the last 12 months, but none
of them had been treated against nosema disease.

The selected colonies were evaluated and sampled in the fall 2007 (late October to mid
November) spring 2008 (late March to mid April), and early summer 2008 (mid to late June) to
obtain individual information of factors that presumably affect colony survivorship and size
of bee populations. On each occasion, the experimental colonies were carefully opened
without using smoke to avoid colony disturbance, and the number of combs covered by bees
were counted to estimate their population (Nasr et al., 1990). After counting frames with bees, samples of ca. 300 workers were collected
from the brood chambers and placed in jars containing 70% ethanol until analyzed. Colonies
were also weighed using a spring scale (Salter 235, London, England; ± 500 g accuracy; 150
kg capacity), and the weight of the equipment and bees were subtracted from the total colony
weight. Weight was used as an indicator of food reserves since most of the weight of
colonies in the fall corresponds to food reserves (Szabo, 1982).

Mean fall, spring, and summer conditions ( ± SE) of honey bee colonies in Ontario,
Canada, for factors that could be associated to colony mortality and low spring and
summer bee populations.

The worker bee samples were analyzed for presence or apparent absence of
tracheal mites, varroa mites and spores of Nosema spp., as well as for
levels of these parasites as per Shimanuki and Knox (2000). Colony die-offs were recorded in the spring and summer, when surviving
colonies were assessed again for the same five factors.

Colonies were subcategorized into two levels: high and low, according to their fall
condition for food reserves (high > 23.6 kg; low < 23.6 kg), bee population
(high ≥ 8 frames with bees; low ≤ 7 frames with bees) and varroa mite levels (high ≥ 5.2;
low ≤ 5.1 mites per 100 bees). High and low values were arbitrarily established by being
either above (for high values) or below (for low values) the means obtained for these
variables from all colonies used in the study (n = 408). It was not
possible to subcategorize colonies according to infestation levels of Acarapis woodi
and infection levels caused by Nosema spp., because of low
parasitic rates and low number of colonies affected by these pathogens in the fall.

Data were analyzed using descriptive statistics, paired comparison tests (Mann-Whitney U
tests to compare data from dead and live colonies; Chi square and contingency table analysis
to compare mortality counts by condition or by region) and Spearman rank correlation
analyses. Data on percentage of varroa and tracheal mite infestation were arcsine
square-root transformed before being subjected to analyses of variance and Fisher’s
protected LSD tests. This transformation was necessary to normalize data before analysis.

3. RESULTS

3.1. Mortality rates and colony conditions

Out of 408 colonies evaluated in the fall 2007, 111 were dead in the spring 2008 (27.2%),
and 19 additional colonies died between the spring and early summer of 2008, for a total
annual mortality rate of 31.9%. On average, colonies lost 42% of their bee populations and
47% of their food reserves during winter. However, colony populations and food reserves
increased 267% and 241%, respectively, by early summer (Tab. I). The infestation levels of varroa mites decreased during the winter
and early summer, whereas tracheal mite infestation levels remained stable throughout the
study period. Conversely, Nosema infection levels increased 317 fold
after the winter to more than 3.1 million spores per bee, but decreased to slightly over
1.1 million spores per bee by early summer (Tab. I).

Colonies that died during the winter had significantly lower bee populations and food
reserves as well as higher mite infestation levels than surviving colonies during the
previous fall (P < 0.001). No differences in Nosema
infection levels were found between colonies that died and those that survived
(Tab. II).

Mean fall conditions ( ± SE) of 408 honey bee colonies found alive or dead the
following spring in Ontario, Canada, for different factors that could be associated
to colony mortality and low spring and summer bee populations. P
based on Mann-Whitney U tests.

3.3. Relative effect of different factors on colony mortality

When examined as a fraction of all morbidity factors, fall varroa mite infestation was
the leading cause of winter colony mortality, followed by fall bee populations and food
reserves (Tab. III). All the factors studied had a
significant effect on colony mortality (P < 0.001), except
Nosema infections.

Effect of different fall factors on winter mortality for 408 honey bee colonies
found alive or dead the following spring in Ontario, Canada. P
based on Chi square tests.

More than 23% of the studied colonies were affected by a single factor, 38.2% by two and
21.8% by three. Varroa mites were part of 11 of the 17 conditions into which colonies were
categorized, which when summed, related to > 85% of the total cases of colony
mortality. The conditions of single or combined factors that showed a higher association
with total colony mortality were: (1) the combination of varroa mite presence, low bee
population and low food reserves (25.2%), (2) the combination of varroa mite and nosema
disease presence, low bee population and low food reserves (12.6%), (3) the combination of
varroa mite presence and low food reserves (10.8%), and (4) the sole presence of varroa
mites (10.8%). Only one entirely unaffected colony (for the factors studied) died out of
21.

4. DISCUSSION

4.1. Mortality rates and colony conditions

The winter mortality rate of the colonies monitored in this study was 27.2%, close to the
32% reported by Ontario beekeepers in a survey (Guzmán-Novoa, 2008). Furthermore, if the
19 additional colonies that died between the spring and early summer 2008 are added to the
equation, the annual mortality rate increases to 31.9%. Our results on colony mortality
thus seem to reflect what occurred across the province of Ontario.

Colonies lost population and weight during winter and gained weight during the spring.
Parasitic mite levels decreased over the winter, likely as a consequence of the lack of
sufficient bee brood to reproduce (in the case of varroa mites) and because of the lack of
young bees to parasitize (in the case of tracheal mites) (Bailey and Ball, 1991). The fact that the percentage of varroa-infested
bees had decreased by the summer may be misleading. It is probable that in the presence of
brood, many mites had been in capped brood cells and actually the mite load per colony had
increased as has been shown in studies on mite population dynamics (Fries et al., 1994; Martin, 1998; Wilkinson and Smith, 2002). The low
parasitic levels of Acarapis woodi and the low number of colonies
infested with this mite could be interpreted, at least partially, as a consequence of the
many years of intensive selection for tracheal mite resistance conducted by beekeepers and
scientists in Ontario (Nasr et al., 2001).

The most striking result regarding variation of the factors studied was the explosive
growth in Nosema infection levels (317-fold) after the winter. This
dramatic increase in Nosema infection in the spring may have occurred
because when colonies recommence their brood production, young bees become infected with
Nosema spores as they clean contaminated comb cells for the queen to
lay eggs (Fries, 1997).

4.2. Colony mortality and factors by region

Varroa mite infestation levels varied significantly among regions and showed a similar
pattern to that of mortality rates. The highest and lowest varroa infestation rates
coincided with the same regions in which colony mortality rates were high and low,
respectively. These results as well as those discussed below, strongly suggest that varroa
mites may be the main cause associated with the mortality of the colonies studied. Ontario
is a representative beekeeping region of northern climates; therefore, the importance that
varroa mites seem to have on the survivorship of overwinterd colonies in Ontario may be
similar in other northern regions of the world.

4.3. Relative effect of different factors on colony mortality

Nosema disease and tracheal mites were apparently the least damaging factors, while
higher fatality cases were associated with varroa mites either alone or in combination
with other factors (> 85%), followed by low bee populations associated to other
factors (> 69%). These results would be expected for varroa mites and for weak
colonies, since it has been shown that these two factors can be directly associated to
overwintered colony losses (Downey et al., 2000).
However, they do not necessarily mean that Nosema and Acarapis
woodi are harmless parasites. Very few colonies were infested with tracheal
mites (only 6.1%), which could have accounted for their low effect on colony mortality in
this study. In the case of nosema disease, although a considerable number of colonies were
Nosema positive (114 colonies out of 408, or 27.9%), these colonies had
low fall infection levels (9804 ± 4688 spores per bee).

The relative weight of nosema disease in the mortality of overwintered colonies is a
controversial matter. Higes et al. (2006, 2007, 2008) and
Martín-Hernández et al. (2007) attribute a major
effect to Nosema infections in the mortality of colonies. However, there
is no conclusive evidence to affirm that Nosema is an important factor in
the recent honey bee die-offs worldwide (Stankus, 2008). Certainly our results do not support the arguments of Higes et al. (2006, 2007,
2008) and Martín-Hernández et al. (2007). It is possible however, that the same honey bee
parasites may cause varying degrees of damage depending on their geographical locations.
It is also possible that other factors not studied could have contributed to the observed
mortality cases, although their contribution would have been insignificant considering
that only one entirely unaffected colony (for the factors studied) died out of 21.

It is relevant to highlight that surviving colonies had mean varroa infestation levels
lower than 3% (2.9 ± 0.2%), and 5% or lower for surviving colonies in the low infested
category during the fall. These fall varroa infestation rates are lower than previously
published acceptable mite loads (Delaplane and Hood, 1997; Currie and Gatien, 2006). Perhaps
acceptable mite loads need to be re-assessed downwards.

4.4. Relative effect of different factors on colony populations

The effect the factors studied had on worker bee life span during winter can be inferred
from the results on bee population in the spring 2008; specifically those from colonies
that were positive to any of the three parasites, or from colonies that had low bee
populations and weight in the fall 2007. Results suggest that varroa mites significantly
reduced colony bee populations, which is in agreement with previous studies (Strange and
Shepard, 2001; Murilhas, 2002; Martin, 2001; Sumpter and
Martin, 2004). Nosema disease and tracheal mites
did not show a significant effect on bee populations, which could be explained by the
reasons discussed above. These results stress the importance of maintaining minimum varroa
mite populations in the fall. Low colony population and food reserves also negatively
affected spring populations of bees, which in turn could have an effect on bee populations
in the summer, and consequently on colony productivity.

Varroa mites, Nosema, and low bee populations in the spring were
associated to slower colony development. Colonies that tested negative or had low levels
of the above parasites, as well as colonies that were strongly populated in the spring,
had significantly more bees in early summer than colonies with high levels of these
pathogens or that were weakly populated in the spring. These results support the
conclusion that varroa mites, nosema disease, and weak spring colony populations,
compromise colony buildup.

The results of this study are not conclusive because the colony conditions for the five
factors analyzed were not controlled. Despite its limitations, field studies like this are
valuable because a large number of colonies were evaluated under natural conditions, as
they were managed by beekeepers, which might shed light on the causes of winter colony
losses. Overall, results suggest that varroa mites could have a high and negative impact
on the survivorship of overwintered honey bee colonies. Moreover, these mites along with
Nosema infections and weakly populated colonies might significantly
restrain the growth of honey bee colonies during the spring in northern climates.

Acknowledgments

Conducting this study was made possible by the efforts of an extensive list of
collaborators. The following people provided substantial contribution: Brian Lacey, Laura
Campbell, Rodrigo León, Raquel Mijares, and Nancy Bradbury sampled colonies and diagnosed
bee diseases. Otillie Welsh provided training in tracheal mite diagnosis. The Technology
Transfer Team of the Ontario Beekeepers’ Association (OBA) and Doug McRory, the provincial
apiarist, provided assistance in countless ways. We are particularly grateful to the
participating beekeepers as well as to the NSERC, the OBA, the IICA, and the Ontario
Ministry of Agriculture, Food and Rural Affairs for funding this study.

Mean fall conditions ( ± SE) of 408 honey bee colonies found alive or dead the
following spring in Ontario, Canada, for different factors that could be associated
to colony mortality and low spring and summer bee populations. P
based on Mann-Whitney U tests.